NKG2D-IG fusion protein for cancer immunotherapy

ABSTRACT

Methods and compositions for cancer immunotherapy are provided. The methods involve the use of a chimeric molecule (e.g., fusion protein) comprising a dimeric NKG2D portion and an Fc portion, which binds one or more NKG2D ligands. In some embodiments, the molecule further comprises a drug moiety (e.g., an IL15/Ra moiety). The methods disclosed herein are useful for the treatment of cancer that is associated with abnormal expression of one or more NKG2D ligands.

RELATED APPLICATIONS

This application is a division of and claims priority under 35 U.S.C. §120 to U.S. patent application Ser. No. 15/775,568, filed May 11, 2018,now U.S. Pat. No. 10,865,232, issued Dec. 15, 2020, which is a nationalstage filing under 35 U.S.C. § 371 of International Application No.PCT/US2016/061479, filed Nov. 11, 2016, which claims priority under 35U.S.C. § 119(e) to U.S. Provisional Application Ser. No. 62/255,016,filed Nov. 13, 2015, the content of each of which is incorporated hereinby reference in its entirety.

BACKGROUND OF INVENTION

NKG2D is a type II transmembrane glycoprotein having an extracellularlectin-like domain. This domain lacks the recognizable calcium-bindingsites found in true C-type lectins and binds protein rather thancarbohydrate ligands. NKG2D is an activating receptor that is expressedin a variety of immune cells. Human NKG2D is expressed on CD8+αβ Tcells, γ6 T cells, NK cells and NKT cells. In mouse systems, NKG2D alsooccurs on macrophages. Human ligands for NKG2D include MHC class Ichain-related molecules (MICA and MICB), UL16-binding proteins (ULBP1,ULBP2, ULBP 3 and ULBP4) and RAET-1G; and mouse ligands for NKG2Dinclude minor histocompatibility antigen 60 (H60) and retinoic acidearly inducible transcript (RAE-1). Expression of NKG2D ligands alsooccurs in intestinal epithelial cells, tumor cells and under conditionsof stress or infection.

NKG2D exists as a disulfide-linked homodimer that delivers an activatingsignal upon ligand binding. Signaling requires association with anadapter protein. Alternative splicing of the NKG2D mRNA results inisoforms with different cytoplasmic domains that can associate eitherwith DAP12 to deliver a true activating signal or with DAP10 resultingin a costimulatory signal. NKG2D has been implicated in immunesurveillance and immune response against viral infection. In addition,elevated levels of NKG2D ligands have been detected in proliferatingcells and many types of cancer.

Certain NKG2D-Fc chimeras and their uses have been disclosed previously,for example in published PCT application WO/2010/080124, the entirecontent of which is incorporated herein by reference.

SUMMARY OF INVENTION

In the present disclosure, novel compositions and methods for cancertherapy are provided. The present invention is based, at least in part,on the surprising discovery that a chimeric molecule comprising twoNKG2D fragments and an Fc fragment (e.g., a dimeric NKG2D-Fc chimera),which is capable of binding one or more NKG2D ligands, induces tumorcell death with improved efficacy compared to chimeric moleculescomprising a single NKG2D fragment and an Fc fragment (e.g., a monomericNKG2D-Fc chimera). In some embodiments, the dimeric NKG2D-Fc chimeradescribed by this document binds with increased avidity to an NKG2Dligand as compared to a monomeric NKG2D-Fc chimera. In some embodiments,the avidity is increased 2-fold, 5-fold, 10-fold, 100-fold, or1000-fold.

Accordingly, in some aspects the disclosure provides a dimeric NKG2D-Fcchimera comprising: NKG2D₁-NKG2D₂-Fc, wherein NKG2D₁ and NKG2D₂ eachcomprises NKG2D or a fragment thereof and can bind an NKG2D ligand; andFc comprises a fragment crystallizable region (Fc) of an immunoglobulin.In some aspects, the disclosure provides a composition comprising thedimeric NKG2D-Fc chimera as described herein and a pharmaceuticallyacceptable carrier.

In some embodiments, the dimeric NKG2D-Fc chimera further comprises adrug moiety. In some embodiments, the drug moiety is attached to theamino terminus or the carboxy terminus of the chimera. In someembodiments, the drug moiety is attached to the carboxy terminus of thechimera.

In some embodiments, the dimeric NKG2D-Fc chimera further comprises atleast one linking molecule, wherein the at least one linking molecule isnot a contiguous portion of the NKG2D₁, NKG2D₂, Fc or drug moiety andwhich covalently joins: an amino acid of NKG2D₁ to an amino acid ofNKG2D₂, an amino acid of NKG2D₂ to an amino acid of Fc, or an amino acidof Fc to the drug moiety.

In some embodiments, the at least one linking molecule is a peptidelinker. In some embodiments, the peptide linker ranges from about 2 toabout 25 amino acids in length. In some embodiments, the at least onelinking molecule is a glycine-serine linker. In some embodiments, theglycine-serine linker is represented by the formula (GS)_(n), wherein nis 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12. In some embodiments, theglycine-serine linker is represented by the formula (GGGGS)_(n) (SEQ IDNO: 2), wherein n is 1, 2, 3, 4, or 5.

In some embodiments, the chimera comprises three linking molecules, X₁,X₂ and X₃, wherein X₁ covalently joins an amino acid of NKG2D₁ to anamino acid of NKGD₂; X₂ covalently joins an amino acid of NKG2D₂ to anamino acid of Fc; and X₃ covalently joins an amino acid of Fc to thedrug moiety. In some embodiments, X₁ is (GS)₃ (SEQ ID NO: 4) and X₂, X₃,and X₄ are each (GGGGS)₄ (SEQ ID NO: 3).

In some embodiments, the NKG2D fragment comprises an extracellularfragment of NKG2D. In some embodiments, the NKG2D extracellular fragmentis represented by SEQ ID NO: 1.

In some embodiments, the Fc comprises a fragment crystallizable region(Fc) of a human immunoglobulin (IgG). In some embodiments, the humanimmunoglobulin is IgG1.

In some aspects, the disclosure provides a method for treating cancercomprising administering to a subject having an NKG2D ligand expressingcancer a dimeric NKG2D-Fc chimera as described by this document in anamount effective to treat the cancer.

In some embodiments, the NKG2D ligand expressing cancer is melanoma,lung cancer, plasma cell cancer, leukemia, lymphoma, ovarian cancer,colon cancer, pancreatic cancer or prostate cancer. In somecircumstances, one or more of these cancers may be present in a subject.

In some embodiments, the method further comprises treating the subjectwith an additional anti-cancer therapy. In some embodiments, theadditional anti-cancer therapy is selected from the group consisting ofsurgery, radiation therapy, chemotherapy, gene therapy, DNA therapy,viral therapy, RNA therapy, adjuvant therapy, and immunotherapy.

In some embodiments, the additional cancer therapy is a chemotherapythat damages DNA.

In some embodiments, the NKG2D ligand is MICA, MICB, ULBP1, ULBP2,ULBP3, ULBP4, ULBP5, or ULBP6.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention. This invention is not limited in its application to thedetails of construction and the arrangement of components set forth inthe following description or illustrated in the drawings. The inventionis capable of other embodiments and of being practiced or of beingcarried out in various ways. Also, the phraseology and terminology usedherein is for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing”, “involving”, and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a diagram of dimeric NKG2D-Fc chimeras with (right) andwithout (left) a drug moiety.

FIG. 2 shows that hNKG2Dx2-hIgG1-hIL15/Ra is produced as a single fusionprotein, and is purified by protein A.

FIG. 3 shows that hNKG2Dx2-hIgG1-IL15/Ra promotes proliferation of humanNK cells similarly to IL-15.

FIG. 4 shows that hNKG2Dx2-hIgG1-IL15/Ra promotes potent killing ofmultiple cell lines, and is superior to hNKG2Dx2-hIgG1 in cell lineswith moderate ligand expression. Panel A shows that neither constructpromotes killing of the B16 tumor cell line, which does not expressNG2D-L. Panel B shows that both constructs equally promote killing of asynthetic B16 tumor cell line expressing high levels of NKG2D ligand.Panel C shows that various tumors express different levels of NKG2Dligands on their cell surface, as measured by NKG2D fusion proteinbinding. Panel D shows that hNKG2Dx2-hIgG1-IL15/Ra kills cellsexpressing moderate NKG2D ligand more efficiently than hNKG2Dx2-hIgG1.

FIG. 5 shows that resting NK cells are activated by the fusion proteinto produce IFN-γ, but maximum production requires all three components:NKG2D, hIgG1, and IL-15. N297Q is a mutation in hIgG1 that prevents CD16(expressed on NK) binding to hIgG1.

FIG. 6 shows that pre-activated NK cells require CD16 binding to killtarget cells, but do not require IL-15.

FIG. 7 shows that optimal activation of, and killing by, resting NKcells requires CD16 binding and IL-15 activation.

FIG. 8 shows ELISA analysis demonstrating that NKG2Dx2-hIgG1 binds toMICA*008 with improved avidity as compared to hNKG2Dx1-hIgG1, which ismonomeric NKG2D-Fc chimera.

FIG. 9 shows flow cytometry analysis demonstrating that hNKG2Dx2-hIgG1binds with improved avidity to NKG2D ligand-expressing cells as comparedto hNKG2Dx1-hIgG1.

FIG. 10 shows NKG2D-Fc drives NK cell killing of ligand-positivetargets. Dimeric NKG2D-Fc chimeras are more effective in mediatingkilling than monomeric NKG2D-Fc chimeras. * depicts p<0.5, and **depicts p<0.01.

FIG. 11 shows dimeric NKG2D-Fc chimeras (e.g., hNKG2Dx2-hIgG1) killNKG2D ligand-expressing cells more efficiently than monomeric NKG2D-Fcchimeras (e.g., hNKG2Dx1-hIgG1). ** depicts p<0.01.

FIG. 12 shows NKG2Dx2-hIgG1 neutralization of soluble MICA is superiorto NKG2Dx1-hIgG1. * depicts p<0.05, ** depicts p<0.01, *** depictsp<0.005, and **** depicts p<0.001.

FIG. 13 shows a structural model of dimeric hNKG2D-hIgG1 in complex withhuman MICA (hMICA). (G₄S)₄ is SEQ ID NO: 3; GGSGGGSG is SEQ ID NO: 5.

DETAILED DESCRIPTION OF INVENTION

Disclosed herein are novel compositions and methods for cancerimmunotherapy. Compositions and methods of the present invention arebased, at least in part, on the surprising discovery that a chimericmolecule comprising two NKG2D fragments and an Fc fragment (e.g., adimeric NKG2D-Fc chimera), which is capable of binding one or more NKG2Dligands, induces tumor cell death with improved efficacy compared tochimeric molecules comprising a single NKG2D fragment and an Fc fragment(e.g., a monomeric NKG2D-Fc chimera).

Monomeric NKG2D-Fc chimeras described in the prior art (e.g., constructsdescribed in published PCT application WO/2010/080124), exhibit a lowbinding avidity to NKG2D ligands (e.g., a low binding avidity index).The dimeric NKG2D-Fc constructs described herein provide increasedbinding avidity (e.g., an improved avidity index of at least 1.1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more) compared to the prior art monomericconstructs by providing multiple NKG2D receptors (or portions thereof)on the same molecule. Without wishing to be bound by any particulartheory, the presence of multiple NKG2D receptors on a single molecule isthought to increase the number and duration of NKG2D-NKG2D ligandbinding interactions, leading to increased anti-tumor activity. Indeed,as shown in the Examples section, dimeric NKG2D-Fc chimeras exhibit upto 100-fold improved binding avidity compared to the prior art monomericNKG2D-Fc chimeras. However, the success of this approach was notpredictable because it was not known whether increasing the number ofreceptors (or portions thereof) in the chimeric construct would inhibitbinding interactions (e.g., via steric hindrance), or cause aggregationof the chimeras that could interfere with the stability of the molecule.

NKG2D ligand(s) are known to be expressed on cancer cells. Therefore, insome embodiments, the disclosure provides methods for cancer therapy ina subject (e.g., a human subject), the method comprising administeringto a subject having an NKG2D ligand-expressing cancer a dimeric NKG2D-Fcchimera as described herein. Unlike an immunotherapy that employs amonoclonal antibody against an NKG2D ligand, such as MICA, the methodsprovided herein are believed to have broad effects against cancer, onthe basis that NKG2D binds to multiple ligands.

The dimeric NKG2D-Fc chimera can target any or all NKG2D ligands thatare expressed on human tumor cells, and thus is capable of mediatingtumor cell destruction through complement lysis and ADCC. The NKG2D-Fcchimera is also capable of opsonizing any tumor cells that express atleast one NKG2D ligand. The NKG2D-Fc chimera can promote efficientcross-presentation (e.g., priming) by dendritic cells, leading to theinduction of potent T cell responses against the tumor. Moreover, thischimera is capable of binding and sequestering any “shed” (e.g., solubleor released) NKG2D ligand(s) produced by tumor cells, therebyalleviating immune suppression due to down-regulation of NKG2Dexpression in response to tumor-derived soluble ligands.

NKG2D-Fc

In some aspects the disclosure provides a dimeric NKG2D-Fc chimeracomprising: NKG2D₁-NKG2D₂-Fc, wherein NKG2D₁ and NKG2D₂ each comprisesNKG2D or a fragment thereof and can bind an NKG2D ligand; and Fccomprises a fragment crystallizable region (Fc) of an immunoglobulin. Insome embodiments, the NKG2D fragment comprises an extracellular fragmentof NKG2D. In some embodiments, the NKG2D extracellular fragment isrepresented by SEQ ID NO: 1.

As used herein, a “dimeric NKG2D-Fc chimera” is a chimeric moleculecomprising two NKG2D ligand binding sites, wherein each ligand bindingsite comprises at least a portion or all of the NKG2D receptor and iscapable of binding an NKG2D ligand. The ligand binding sites are fusedto an Fc fragment. In the Examples section and the Figures, the twoNKG2D ligand binding sites of dimeric NKG2D-Fc chimera are also referredto collectively as “NKG2Dx2”. The monomeric NKG2D-Fc chimera describedin the prior art can be referred to as “NKG2Dx1”. The terms “chimera,”“chimeric molecule,” and the like generally refer to a molecule that iscomprised of parts that are from multiple origins or sources. In someembodiments, dimeric NKG2D-Fc is produced as a recombinant chimericfusion protein.

In some embodiments, the dimeric NKG2D-Fc chimera described herein bindswith increased avidity to an NKG2D ligand as compared to a monomericNKG2D-Fc chimera. As used herein, “avidity” refers to overall strengthacross multiple affinities of individual non-covalent bindinginteractions between a ligand and a receptor. Methods of measuringbinding avidity are known in the art and include, for example, ELISA,surface plasmon resonance analysis, CD analysis, fluorescence quenching,size-exclusion binding assay and isothermal titration calorimetry. Forbrief descriptions of these assays, see, for example, Lengyel et al.,2007, J. Biol. Chem., 282: 30658-666). In some embodiments, bindingavidity is determined by measuring avidity index. In some embodiments,the binding avidity of the dimeric NKG2D-Fc chimera to a NKG2D ligand isincreased between about 2-fold and about 2000-fold as compared to themonomeric NKG2D-Fc chimera. In some embodiments, the binding avidity isincreased between about 2-fold and 1000-fold. In some embodiments, thebinding avidity is increased between about 2-fold and 100-fold. In someembodiments, the binding avidity is increased between about 5-fold and1000-fold. In some embodiments, the binding avidity is increased betweenabout 5-fold and 200-fold. In some embodiments, the binding avidity isincreased between about 2-fold and about 20-fold. In some embodiments,the binding avidity is increased 2-fold, 5-fold, 10-fold, 100-fold, or1000-fold. In some embodiments, the binding avidity is increased atleast 2-fold, at least 5-fold, at least 10-fold, at least 100-fold, orat least 1000-fold. In some embodiments, the dimeric NKG2D-Fc constructshas an increased binding avidity index as compared to the monomericNKG2D-Fc chimera, e.g., an improved avidity index of at least 1.1, 2, 3,4, 5, 6, 7, 8, 9, 10 or more.

NKG2D

In some aspects the disclosure provides a dimeric NKG2D-Fc chimeracomprising: two NKG2D or fragments thereof. NKG2D, also referred to asKLRK1; killer cell lectin-like receptor subfamily K, member 1; CD314;KLR; NKG2-D; FLJ17759; F1175772 or D12S2489E, is one of the majortriggering receptors of NK cells and is well known in the art. See, forexample, Garrity et al. (2005). The portion of the NKG2D receptor usedfor dimeric NKG2D-Fc is based on the known sequences of NKG2D (e.g.,Accession: NP_031386) or derivatives thereof that bind at least oneligand. Derivatives of NKG2D that can be used in the compositions andmethods of the invention include, but are not limited to, NKG2Dsequences containing one or more mutations, such as a point mutation, asubstitution, a deletion mutation and/or an insertion mutation. One ofordinary skill in the art can readily determine suitable derivatives ofNKG2D according to the teaching of the present disclosure and knowledgeavailable in the art. At the cDNA level, such a mutation may be a silentmutation. Alternatively, the mutation may result in a change in thecorresponding amino acid residue. Where the latter is the case, thechange may constitute a conservative change, such that an amino acidresidue is replaced with another amino acid residue of similarcharacteristics. In some cases, however, a mutation may result in asubstitution that is non-conservative. Such mutations are acceptable tothe extent that the dimeric NKG2D-Fc chimera is capable of binding to anNKG2D ligand.

In some embodiments, each NKG2D portion of a dimeric NKG2D-Fc chimera isa full length NKG2D polypeptide. The full length sequence of NKG2D hasbeen described in the literature. See, for example, RefSeq Accession:NP_031386. Additionally, alternative splice variants of NKG2D have beendescribed. For purposes of the instant invention, any one of suchalternatively spliced variants may be used, provided that the resultingpolypeptide, when constructed as a dimeric NKG2D-Fc chimera, is capableof binding its ligand(s).

In some embodiments, each NKG2D portion of a dimeric NKG2D-Fc chimera isa partial sequence (i.e., fragment) of the NKG2D receptor polypeptide,provided that the resulting polypeptide, when constructed as a dimericNKG2D-Fc chimera, retains the ability to bind its ligand(s). Forexample, each NKG2D portion of the dimeric NKG2D-Fc construct may beshortened by either end of the NKG2D sequence by one or more amino acidresidues. More specifically, the N-terminus of the NKG2D sequence may bedeleted by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, about 30, about 40, about 50, about 60, about 70, about 80or more residues. Similarly, the C-terminus of the NKG2D sequence may bedeleted by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20 or more residues. In some embodiments, both the N-terminusand the C-terminus may be shortened as described.

It has been shown that the extracellular portion of NKG2D contributes tothe formation of homodimers and forms a ligand-binding site(s). Thus, itis possible to delete part or all of the intracellular portion of NKG2Dand still maintain the ability to bind its ligand(s). For example, thedimeric NKG2D-Fc chimera described in this disclosure may containpredominantly an extracellular fragment of the NKG2D receptor.Structural analyses have revealed that amino acid residues 78 to 216 ofthe human NKG2D sequence correspond to the extracellular portion of theNKG2D, containing ligand-binding sites. For a murine counterpart, theextracellular domain is amino acid residues 78-232, 94-232 or 92-232.

Accordingly, in some embodiments, each NKG2D of the dimeric NKG2D-Fcconstruct comprises the extracellular portion of the NKG2D sequence,e.g., amino acid residues 78-216 of human NKG2D; 78-232, 94-232 or92-232 of murine NKG2D. In some embodiments, a dimeric NKG2D-Fcconstruct comprises a portion of the extracellular domain. Thus, theextracellular domain of the dimeric NKG2D-Fc construct may be shortenedat the N-terminus, at the C-terminus, or both. For example, theN-terminus of the extracellular domain used to generate a dimericNKG2D-Fc may be shortened by one or more amino acid residues, e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,about 30, about 40, about 50, about 60, and so forth, relative to thefull extracellular portion of the polypeptide. The C-terminus of theextracellular domain used to generate an NKG2D-Fc may be shortened byone or more amino acid residues, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, about 30, about 40, about 50,about 60, and so forth, relative to the full extracellular portion ofthe polypeptide. Using a human NKG2D as an example, the dimeric NKG2D-Fcconstruct may contain a fragment of the extracellular domain, whereinthe N-terminus of the domain begins at amino acid residue 79, 80, 81,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, about 110, about 120, about130, about 140 or about 150. Similarly, the dimeric NKG2D-Fc constructmay contain a fragment of the extracellular domain, wherein theC-terminus of the domain ends at amino acid residue 231, 230, 229, 228,227, 226, 225, 224, 223, 222, 221, 220, 219, 218, 217, 216, 215, 214,213, 212, 211, 210, 209, 208, 207, 206, 205, and so forth. Suchdeletions at each end of the extracellular domain of the NKG2D sequencemay be combined.

The skilled artisan recognizes that dimeric NKG2D-Fc chimera describedby the disclosure may comprise two of the same NKG2D fragments or twodifferent NKG2D fragments. For example, in some embodiments, a dimericNKG2D-Fc chimera comprises two NKG2D fragments corresponding to aminoacid residues 78 to 216 of the human NKG2D. In some other embodiments, adimeric NKG2D-Fc chimera comprises two NKG2D fragments, where the firstfragment corresponds to amino acid residues 78 to 216 of the human NKG2Dand the second fragment corresponds to a different portion of the NKG2Dextracellular domain (e.g., amino acid positions 140 to 210 of the humanNKG2D).

Also contemplated are dimeric NKG2D-Fc derivatives that include one ormore mutations in the NKG2D portion of the construct at the interface ofthe NKG2D-ligand binding. In particular, certain mutations are known toaffect the binding affinity between the NKG2D receptor and its ligand(e.g., MICA). See, for example, Lengyel et al., 2007, J. Biol. Chem.,282: 30658-666. The three dimensional structure of a complex betweenNKG2D and MICA has been described. Accordingly, one of ordinary skill inthe art may determine the amino acid residues of NKG2D that contributeto the interaction with its ligand and test the effect of mutations bysystematically altering the key residues. In any of the embodiments, theresulting dimeric NKG2D-Fc chimera is capable of binding ligand(s). Fora comprehensive review of the amino acid residues that are involved inreceptor-ligand contact, see, for example, Strong and McFarland, 2004,Advances in Protein Chemistry, 68: 281-213. According to publishedstudies, key residues that are thought to be important in theinteraction with the ligand have been mapped to amino acid residuesapproximately from 150 to 207 in human NKG2D, which correspond toresidues approximately from 166 to 223 in mouse NKG2D. Therefore, eachNKG2D fragment of the dimeric NKG2D-Fc construct of the inventionpreferably comprises a fragment spanning at least most of these residues(e.g., residues 150 to 207 in human NKG2D). Likewise, it will beunderstood that conservative substitutions, deletions or mutationsoutside these regions can potentially be tolerated with ease in manyinstances.

Some amino acid residues have been identified to be especially importantfor mediating ligand binding. Specifically, residues of human NKG2Dimportant for binding to MICA include Y152, Q185, K197, Y199, E201 andN207. Residues of human NKG2D important for binding to ULBP3 include1182, Y199 and Y152. Residues of murine NKG2D important for binding toRAE-1β include K166, Y168, Y215, K213, E217 and N223. In preferredembodiments, therefore, most or all of these residues (of acorresponding dimeric NKG2D construct) are maintained without a mutationor deletion at the position where broad permissibility (e.g.,specificity) for multiple ligands is desirable. However, it is alsopossible to design a dimeric NKG2D-Fc construct that preferentiallybinds one ligand over another ligand by strategically introducing amutation at one or more of these key residues that confer selectiveligand-recognition and binding. On the other hand, certain amino acidresidues are involved in the binding of various ligands. For example,Y152 and Y199 in human NKG2D, which are equivalent to Y168 and Y215respectively in the murine counterpart, contribute to the binding ofMICA as well as ULBP3. Therefore, in some embodiments, these residuesare unmodified so as to retain broad ligand specificity.

The Examples provided below present a representative dimeric NKG2D-Fcchimera, wherein each NKG2D fragment corresponds to amino acid residues78 to 216 of the human NKG2D. However, it should be appreciated that thesame approach may be employed for NKG2D sequences derived from any otherspecies that are known to develop cancer. For example, the NKG2Dfragment of dimeric NKG2D-Fc may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 ormore amino acid changes, such as deletions, insertions andsubstitutions, as long as the dimeric NKG2D-Fc retains its ligandbinding activity.

The present invention includes variants of dimeric NKG2D-Fc constructsthat contain one or more amino acid changes as described above, to theextent that the dimeric NKG2D-Fc chimera binds to its native ligand orligands. To determine whether a dimeric NKG2D-Fc variant containing aparticular mutation retains ligand binding activity, binding assays canbe carried out, in which binding affinity and/or binding capacity of theparticular dimeric NKG2D-Fc chimera to its ligand(s) may be evaluated. Anumber of methods are known in the art by which receptor-ligandinteractions may be measured. These methods for assaying ligand bindinginclude, without limitation, ELISA, surface plasmon resonance analysis,CD analysis, fluorescence quenching, size-exclusion binding assay andisothermal titration calorimetry. For brief descriptions of theseassays, see, for example, Lengyel et al. (2007).

Fc Fragment

In some embodiments, a dimeric NKG2D-Fc chimera comprises a fragmentcrystallizable region (Fc) of an immunoglobulin. The Fc region ofimmunoglobulins plays a significant role in mediating immune defense.FcγRs are widely expressed as transmembrane glycoproteins on a number ofcell types, including macrophages, NK cells, dendritic cells, B cells,neutrophils and mast cells. Fc-mediated activities include recruitmentof effector cells via Fc-FcγR interactions. There are two classes of Fcreceptors that can be distinguished functionally: the activating Fcreceptor class and the inhibitory Fc receptor class. Activating Fcreceptors include human FcγRIA, FcγRIIA and FcγRIIIA, as well as theirmurine orthologues, i.e., FcγRI, FcγRIII FcγRIV. Activating FcγRsmediate ADCC and ADCP, induce endocytosis of immune complexes leading toantigen presentation, and contribute to the production and release ofcytokines and proinflammatory factors. For general review of the IgGstructure and mechanisms of action, see Liu et al. (2008; ImmunologicalReviews, 222: 9-27). As described in more detail herein, the Fc portionof dimeric NKG2D-Fc is a domain that binds an activating Fc receptor,and preferably an activating Fc Ig domain and includes the hinge regionthat allows for dimerization.

The Fc portion of the dimeric NKG2D chimera useful for this disclosurecan be readily adapted to render it species-specific. For use in amurine system, e.g., cells derived from a mouse, the Fc fragment used togenerate dimeric NKG2D-Fc is preferably that of a murine origin. In someembodiments, an Fc fragment of the murine IgG2a is preferred.

For use in a human subject, e.g., for cancer treatment, the Fc fragmentused to generate dimeric NKG2D-Fc is preferably that of a human origin.In particularly preferred embodiments, NKG2D-Fc comprises an activatingFc Ig domain. Among the four human IgG isotypes, an activating Fc domainof IgG1 is preferred for the preparation of dimeric NKG2D-Fc. Thus, insome embodiments, the Fc comprises a fragment crystallizable region (Fc)of a human immunoglobulin (IgG). In some embodiments, the humanimmunoglobulin is IgG1. Experimental data relating to chimericconstructs containing an Fc region of the human IgG1 are provided in theExamples section.

The art appreciates that different antibody isotypes have a varyingdegree of cytotoxic potential in vivo (See, for example, Nimmerjahn F. &Ravetch J V., 2006, Immunity, 24:19-28). For example, the murine IgG2aand IgG2b isotypes are more efficient in clearing infections such asbacterial infections and viral infections and in killing tumor cellsthan their IgG1 or IgG3 counterparts. This is attributable at least inpart to differential ratios of activating versus inhibitory FcRs presentin vivo. Similarly, with respect to human IgG isotypes, IgG1 and IgG3have a stronger interaction with FcRs than IgG2 or IgG4. Moreover,certain polymorphic allotypes of a given isotype may influence affinityfor an Fc receptor. Indeed, there are allelic variants of activatingFcRs that will significantly affect the affinity for certain antibodyisotypes. For example, the FcγRIIIa receptor 158V allotype displays ahigher affinity for human IgG1 and increased antibody-dependent cellularcytotoxicity (Cartron G. et al., 2002, Blood, 99: 754-758).

Without wishing to be bound by any particular theory, it is possible tooptimize the interaction between the Fc portion of the dimeric NKG2D-Fcchimera to its corresponding Fc receptor by strategically selecting ormodifying the Fc allele used for preparing the dimeric NKG2D-Fc chimera.Accordingly, the invention contemplates using a mutant or an allotype ofan Fc fragment. A number of useful mutations within an Fc domain havebeen described, which can affect the interaction of an Fc and itsreceptor, the effector function of the Fc, as well as the half-life ofthe Fc-containing molecule. These include specific amino acidsubstitutions and/or modifications to carbohydrate moieties in the Fc.For review, see, for example, Liu et al., 2008, Immunological Reviews,222:9-27; Nimmerjahn & Ravetch, 2007, Curr. Opin. Immunol., 19(2):239-45.

The structure of Fc fragments generally is known in the art. Briefly,the Fc region of a typical IgG molecule is a symmetric homodimer of thecarboxy-terminal portion of heavy chains and is composed of the C_(H)2and C_(H)3 domains, which are separated from the Fab by a flexible hingeregion. The Fc region is stabilized by non-covalent interactions betweendomains. The Fc region interacts with FcRs to exert effector functionsor to regulate the catabolism of IgG. The heavy constant regions (Cγ2and Cγ3) and the hinge region located between the variable domain andthe constant regions interact with C1q and Fc receptors (FcRs). Thus,the heavy constant regions of the IgG molecule are responsible for itseffector functions, since they include binding sites for complement andfor FcRs on different effector cells. Recruitment of effector cells istherefore mediated via the Fc-FcγR interactions.

In general, the interaction of an antibody with complement initiatescomplement-dependent cytotoxicity (CDC), and FcγR interactions mediateantibody-dependent cell toxicity (ADCC) and antibody-dependent cellphagocytosis (ADCP). The classical activation pathway of CDC istriggered when C1, the first component of the pathway, binds to thehinge-Fc portion of the IgG in an antigen-antibody complex. Subsequentactivation of the complement cascades eventually induces the formationof a C5-C9 membrane attack complex that leads to the death of the targetcell. ADCC, on the other hand, is dependent upon the ability of theFcγR-bearing cells of the innate immune system (e.g., NK cells,monocytes, macrophages and granulocytes) to recognize the Fc domain ofantibody bound to target cells. This recognition triggers effector cellsto release cytoplasmic perforin, granulysin, and granzymes that induceapoptosis and lysis of target cells. The major effector cells in ADCCare NK cells, which express the type of FcγRs that recognize the IgG1and IgG3 subclasses and trigger cytotoxic effects in vivo.

In the context of the present invention, as demonstrated in theExamples, the dimeric NKG2D-Fc chimeras described herein are capable ofmediating equivalent cellular effects by virtue of having a functionalFc portion, coupled with the dimeric NKG2D portion that can broadly butspecifically recognize and bind to its ligands.

As noted, there are activating receptors (FcγRI, FcγRIIA and FcγRIII)and inhibitory (FcγRIIB) receptors. In general, interaction of IgGs withactivating FcγRs triggers cell activation, while interaction withFcγRIIB inhibits cell activation. With the exception of B cells and NKcells, activating and inhibitory FcγRs are co-expressed on the sameeffector cells, thereby generating a threshold for cell activation. Bcells express only the inhibitory FcγRIIB and therefore cannot beactivated by endogenous IgG under physiological conditions. NK cellsexpress the activating FcγRIII so that they can kill target cellsindependently of pre-activation (or priming).

FcγRIIA and FcγRIII (CD16) have low affinities for monomeric IgG and arethought to be critical for triggering effector functions, leading toanti-tumor activity. Thus, it is possible to design a dimeric NKG2D-Fcsuch that it is genetically engineered to have increased affinities forthe activating FcγRIII, and decreased affinities for the inhibitoryFcγRIIB

Accordingly, the amino acid residues of dimeric NKG2D-Fc molecules thatcontribute to their direct interaction with FcγRs, which are locatedprimarily in the lower hinge region and are adjacent to the Cγ2 region,may be modified, and such variants are embraced by this invention. Ithas been shown that the region corresponding to amino acid residues234-237 of the IgG is required for binding to FcγRs. In addition, otherresidues that are important in IgG-FcγRs interactions have been shown tobe located in the Cγ2 domain and include Asp265, Asp270, Ala327, Pro329and Lys338.

Several strategies are contemplated to generate dimeric NKG2D-Fcchimeras with enhanced activities. To engineer the dimeric NKG2D-Fc withan enhanced ADCC capability, at least two approaches are contemplated.First, based on the amino acid residues in an IgG1 that were identifiedto be critical for its binding to activating and inhibitory FcγRs, theinvention provides variants of dimeric NKG2D-Fc chimeras that enhance orreduce, respectively, the affinity for these receptors. Accordingly, inone embodiment, the triple amino acid substitution,Ser298Ala/Glu333Ala/Lys334Ala, where the position of each residue isbased on IgG1, is provided. The dimeric NKG2D-Fc containing this triplemutation should exhibit a higher affinity for FcγRIIIA but not forFcγRIIB, thereby promoting ADCC. Similarly, in another embodiment, thedimeric NKG2D-Fc variant contains the double mutation in the Fc,Ser239Asp/Ile332Glu, which is expected to exert improved ADCC. Othermutations for enhancing ADCC include, without limitation,Ser239Asp/Ala330Leu/Ile332Glu and Ser239Asp/Ser298Ala/Ile332Ala.Similarly, in some embodiments, mutations that combine increased bindingto FcγRIIIA (e.g., activating receptors) and reduced binding to FcγRIIBare contemplated. Examples of such Fc mutations includePhe243Leu/Arg292Pro/Tyr300Leu/Val305Ile/Pro396Leu, without limitation(the positions of the residues are based on IgG1).

The second approach relates to modifying the carbohydrate moieties inthe Fc based on the observation that some modifications significantlyaffect the affinity of the Fc for FcγRs. It has been shown that the Fcdomain contains two asparagine N-linked oligosaccharide sites (reviewedin Liu et al., 2008). ADCC requires the presence of certainoligosaccharides and is dependent upon changes in the structure of theoligosaccharides. In particular, previous studies have shown thatremoving the fucose moiety attached to the innermost GlcNAc of thebiantennary complex-type oligosaccharides dramatically increases ADCC byimproving the binding of the Fc to FcγRIIIA without impairing CDCactivity. Based on this observation, in one embodiment, the inventionprovides fucose-deficient dimeric NKG2D-Fc. In some embodiments, thechimera completely lacks the fucose moiety (i.e., non-fucosylated). Inother embodiments, the chimera is hypofucosylated.

To make dimeric NKG2D-Fc containing modified carbohydrates, host cellsmay be engineered to express the enzymes that catalyze the desiredmodification(s). For example, host cells, such as Chinese hamster ovary(CHO) cells may be transfected with the enzyme,β-(1,4)-N-acetylglucosaminyltransferase III (GnT-III), which elevatesthe level of bisected, non-fucosylated oligosaccharides. The NKG2D-Fcproduct generated from these host cells can have a dramatically enhancedADCC activity. In addition, in some embodiments, the content of fucosein NKG2D-Fc may be manipulated by α-1,6-fucosyltranferase(FUT8)-knockout cells lacking core-fucosyl transferase activity.Alternatively, small interfering RNA may be used to constitutivelyinhibit the expression of the FUT8 enzyme to achieve the same effect. Insome embodiments, host cells deficient in guanosine diphosphate(GDP)-mannose 4,6-dehydratase (GMD) may be used to yield non-fucosylatedNKG2D-Fc.

Next, to engineer the dimeric NKG2D-Fc with an enhanced complementactivity, various mutations in the Fc domain are contemplated.Generally, complement can be activated by at least three pathways,leading to the formation of the membrane attach complex C5b-9, whichforms pores in the plasma membranes of target cells and causes theirlysis. C1q binding to the Fc domain is a critical step in this process.Among the human IgG subclasses, only IgG1 and IgG3 can initiate thecomplement cascade. In some embodiments, mutations are introduced to theFc domain of the dimeric NKG2D-Fc, so as to promote C1q recruitment andthe C1q-Fc interaction. The residues of the Fc targeted for suchmutations include, but are not limited to: Asp270, Lys322, Pro329 andPro331. These mutations involve substituting the correspondingresidue(s) with nonpolar neutral amino acids, such as Ala, Met, or Trp.In a specific embodiment, the dimeric NKG2D-Fc contains the mutation,Lys326Trp, Clu333Ser or both.

To achieve increased C1q binding and enhanced CDC, some embodiments ofthe invention involve introducing a mutation or mutations to certainresidues of the hinge region of human IgG1. Non-limiting examples ofsuch mutations include: Lys222Trp/Thr223Trp, Cys220Asp/Asp221Cys,Cys220Asp/Asp221Cys/Lys222Trp/Thr223Trp, Lys222Trp/Thr223Trp/His224Trpand Asp221Trp/Lys222Trp.

In addition, it should be noted that when fusion proteins withartificial sequences and activities are used as therapeutic agents, insome circumstances, patients treated with such a fusion protein triggeran unwanted immune response, such as development of antibodies againstthe agent. Certain structural modifications of an Fc fragment have beenshown to reduce immunogenicity of a therapeutic fusion protein. See, forexample, U.S. Pat. No. 6,992,174 B2 by Gillies et al., which isincorporated by reference herein; Liu et al., 2008, ImmunologicalReviews, 222:9-27. Such modifications may be useful for an effectivedesign of dimeric NKG2D-Fc described in the present disclosure.

Linkers

The dimeric NKG2D-Fc construct used in the methods of the presentdisclosure may further comprise at least one linking moiety thatconnects a first NKG2D portion (e.g., NKG2D₁) with a second NKG2Dportion (e.g., NKG2D₂), an NKG2D portion (e.g., NKG2D₁ or NKG2D₂) withan Fc fragment, and/or an Fc fragment to a drug moiety. In someembodiments, a linking moiety (e.g., linking molecule) is referred to asX₁, X₂, or X₃. In some cases, a hinge region of Fc fusion proteinmolecules serves as a spacer between the Fc region and the fused peptide(e.g., soluble receptor), allowing these two parts of the molecule tofunction separately (see, for example, Ashkenazi et al., 1997).

In some embodiments, the at least one linking moiety (e.g., linkingmolecule) is not a contiguous portion of the NKG2D₁, NKG2D₂, Fc, or drugmoiety and covalently joins: an amino acid of NKG2D₁ to an amino acid ofNKG2D₂, an amino acid of NKG2D₂ to an amino acid of Fc, or an amino acidof Fc to the drug moiety. As used herein, a linking molecule that is“not a contiguous portion” means that the each NKG2D portion (e.g.,NKG2D₁ and NKG2D₂), a NKG2D portion and the Fc portion, and/or the Fcportion and a drug moiety of the chimera are connected via an additionalelement that is not a part of the NKG2D or immunoglobulin or drugmoiety, that is contiguous in nature with the portions of the chimerathat it joins, and functions as a linker. Non-limiting examples of alinking molecule that is not a contiguous portion of either NKG2D, Fc,or drug moiety are described below.

The linking molecule may be a peptide linker. In some embodiments, thepeptide linker ranges from about 2 to about 25 amino acids in length. Insome embodiments, the peptide linker is 20 amino acids in length. Insome embodiments, the peptide linker ranges from about 4 to about 16amino acids in length. In some embodiments, the peptide linker is 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25 amino acids in length. In some embodiments, the peptidelinker is longer than 25 amino acids in length. Where the linker is apeptide linker, the dimeric NKG2D-Fc chimera may be produced as a singlerecombinant polypeptide using a conventional molecularbiological/recombinant DNA method.

In some embodiments, a peptide linker provides a protease-dependentcleavable site. Examples of protease-cleavable peptide linkers include,without limitation, the MMP sensitive linker GGPLGLWAGG (SEQ ID NO: 6)and the factor Xa-sensitive linker IEGR (SEQ ID NO: 7). The art isfamiliar with a variety of cleavable sequences that may be employed forthe methods provided herein, for example those disclosed in Chen et al.,Adv. Drug Deliv. Rev. (2013), 65(10): 1357-69).

In some embodiments of the present invention, a flexible peptide linkeris used. A flexible peptide linker is preferably about 25 or fewer aminoacids in length. In some embodiments, a flexible peptide linker is 20amino acids in length. In some embodiments, a peptide linker containsabout 20 or fewer amino acid residues, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, and 20. In some embodiments, apeptide linker contains about 12 or fewer amino acid residues, e.g., 3,4, 5, 6, 7, 8, 9, 10, 11, and 12. In some cases, a peptide linkercomprises two or more of the following amino acids: glycine, serine,alanine, and threonine. In some embodiments, the flexible peptide linkeris a glycine-serine linker.

In some embodiments, the glycine-serine linker is represented by theformula (GS)_(n), wherein n is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.In some embodiments, the glycine-serine linker is represented by theformula (GGGGS)_(n) (SEQ ID NO: 2), wherein n is 1, 2, 3, 4, or 5.

In some embodiments, a dimeric NKG2D-Fc chimera comprises three linkingmolecules, X₁, X₂ and X₃, wherein X₁ covalently joins an amino acid ofNKG2D₁ to an amino acid of NKGD₂; X₂ covalently joins an amino acid ofNKG2D₂ to an amino acid of Fc; and X₃ covalently joins an amino acid ofFc to a drug moiety. In some embodiments, X₁ is (GS)₃ (SEQ ID NO: 4) andX₂, X₃, and X₄ are each (GGGGS)₄ (SEQ ID NO: 3).

In some embodiments, the dimeric NKG2D-Fc chimera contains an IEGR (SEQID NO: 7) peptide linker.

Alternatively, a linking molecule may be a non-peptide linker. As usedherein, a “non-peptide linker” is a biocompatible polymer including twoor more repeating units linked to each other. Examples of thenon-peptide polymer include but are not limited to: polyethylene glycol(PEG), polypropylene glycol (PPG), co-poly (ethylene/propylene) glycol,polyoxyethylene (POE), polyurethane, polyphosphazene, polysaccharides,dextran, polyvinyl alcohol, polyvinylpyrrolidones, polyvinyl ethylether, polyacrylamide, polyacrylate, polycyanoacrylates, lipid polymers,chitins, hyaluronic acid, and heparin. For more detailed descriptions ofnon-peptide linkers useful for Fc fusion molecules, see, for example,WO/2006/107124, which is incorporated by reference herein. Typicallysuch linkers will have a range of molecular weight of from about 1 kDato 50 kDa, depending upon a particular linker. For example, a typicalPEG has a molecular weight of about 1 to 5 kDa, and polyethylene glycolhas a molecular weight of about 5 kDa to 50 kDa, and more preferablyabout 10 kDa to 40 kDa.

Drug Moieties

In some embodiments, a dimeric NKG2D-Fc chimera further comprises a drugmoiety. As used herein, “drug moiety” refers to a therapeutic agent thatis intended for delivery to a targeted cell (e.g., a cancer cell).Generally, a drug moiety is conjugated (e.g., directly or indirectlycovalently bound) to the carboxy terminus of a dimeric NKG2D-Fc chimera.However, the skilled artisan recognizes that in some embodiments, a drugmoiety is conjugated to the amino terminus of a dimeric NKG2D-Fcchimera. Examples of “drug moieties” include drugs (e.g., smallmolecules), toxins (e.g., molecules of the lymphotoxin family),radionuclides, enzymes, cytokines, chemokines, antibody single chainvariable fragments directed against activating compounds or blockingangiogenesis, or essentially any anti-tumor compound.

In some embodiments, the drug moiety comprises a cytokine or functionalportion thereof. Cytokines are proteins and peptides that are capable ofmodulating immune cell function. A “functional portion” of a cytokine isa cytokine fragment that retains the ability to modulate immune cellfunction (e.g., bind to one or more cytokine receptors). Examples ofcytokines include, but are not limited to interferon-alpha (IFN-α),interferon-beta (IFN-β), and interferon-gamma (IFN-γ), interleukins(e.g., IL-1 to IL-29, in particular, IL-2, IL-5, IL-6, IL-7, IL-10,IL-12, IL-15 and IL-18), tumor necrosis factors (e.g., TNF-alpha andTNF-beta), erythropoietin (EPO), MIP3a, monocyte chemotactic protein(MCP)-1, intracellular adhesion molecule (ICAM), macrophage colonystimulating factor (M-CSF), granulocyte colony stimulating factor(G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF).In some embodiments, the drug moiety comprises a cytokine selected fromthe group consisting of: IL-2, IL-12, IL-15, IL-18, IL-21 and IFN-α.

In some embodiments, a drug moiety comprises a cytokine/cytokinereceptor heterocomplex. Cytokine/cytokine receptor heterocomplexes areknown in the art and are described, for example in Rowley et al., Eur JImmunol. 2009 February; 39(2): 491-506. In some embodiments, a dimericNKG2D-Fc chimera includes a drug moiety comprising an IL-15 (e.g.,GenBank AAX37025)/IL-15Ra (e.g., GenBank AAP69528.1) heterocomplex. Insome embodiments, the drug moiety comprises amino acids 31-107 of thehuman IL-15 receptor alpha (hIL15Ra, GenBank AAP69528.1) fused to aminoacids 22-135 of IL-15 (GenBank AAX37025). In some embodiments, the IL-15and IL-15Ra are separated by a linker, for example, a 20-amino acid(G₄S)₄ (SEQ ID NO: 3) linker. A dimeric NKG2D-Fc chimera comprising anIL-15/IL-15Ra heterocomplex is further described in the Examplessection. In some embodiments, a dimeric NKG2D-Fc chimera includes a drugmoiety comprising a heterocomplex of IL-12p35 and IL-12p40. In someembodiments, the IL-12p35 and IL-12p40 are separated by a linker, forexample, a 20-amino acid (G₄S)₄ (SEQ ID NO: 3) linker. In someembodiments, a dimeric NKG2D-Fc chimera includes a drug moietycomprising a heterocomplex of IL-23p19 and IL-23p40. In someembodiments, the IL-23p19 and IL-23p40 are separated by a linker, forexample, a 20-amino acid (G₄S)₄ (SEQ ID NO: 3) linker. In someembodiments, a dimeric NKG2D-Fc chimera includes a drug moietycomprising a heterocomplex of IL-27p28 and EB1. In some embodiments, theIL-27p28 and EB1 are separated by a linker, for example, a 20-amino acid(G₄S)₄ (SEQ ID NO: 3) linker. In some embodiments, each subunit of acytokine/cytokine receptor heterocomplex is on a different chain of thedimeric NKG2D-Fc chimera.

In some embodiments, the drug moiety is an antibody single chainvariable fragment (ScFv). As used herein, an “antibody single chainvariable fragment” refers to a fusion protein of the variable regions ofthe heavy (VH) and light chains (VL) of immunoglobulins, connected witha short linker peptide. ScFv proteins retain the specificity of theoriginal immunoglobulin, despite removal of the constant regions and theintroduction of the linker. In some embodiments, a ScFv binds to animmune checkpoint protein (e.g., PD1 or CTLA4). In some embodiments, anScFv blocks angiogenesis (e.g., binds to a regulator of angiogenesis,such as VEGF).

In some embodiments, the drug moiety is a chemokine. As used herein,“chemokines” refers to low-molecular-weight proteins that stimulaterecruitment of leukocytes. Generally, chemokines are secondarypro-inflammatory mediators that are induced by primary pro-inflammatorymediators such as interleukin-1 (IL-1) or tumor necrosis factor (TNF).Chemokines can be classified into four families: CC chemokines (e.g.,CCL1 to CCL-28), CXC (e.g., CXCL1 to CXCL17), C (e.g., XCL1, XCL2), andCX3C (CX3CL1).

In some embodiments, the drug moiety is a small molecule. As usedherein, “small molecule” refers to a non-peptidic, non-oligomericorganic compound either synthesized in the laboratory or found innature. Non-limiting examples of small molecule drugs include smallmolecule kinase inhibitors (e.g., everolimus, gefitinib, imatinib,etc.), bromodomain inhibitors (e.g., JQ1, I-BET 151, RVX-208, etc.),antibiotics (e.g., kanamycin, neomycin, ciprofloxacin, etc.), andantivirals (e.g., ribavirin, rimantadine, zidovudine, etc.). In someembodiments, the small molecule is an anti-tumor compound. Anti-tumorcompounds are discussed in further detail elsewhere in this disclosure.

In some embodiments, the drug moiety is a radionuclide. As used herein,“radionuclide” refers to medically useful radionuclides. Examples ofradionuclides include ^(99m)Tc, ¹⁸⁸Re, ¹⁸⁶Re, ¹⁵³Sm, ¹⁶⁶Ho, ⁹⁰Y, ⁸⁹Sr,⁶⁷Ga, ⁶⁸Ga, ¹¹¹In, ¹⁸³Gd, ²²⁵Ac, ²¹²Bi, ²¹¹At, ⁴⁵Ti, ⁶⁰Cu, ⁶¹Cu, and⁶⁷Cu.

Other Moieties

In some embodiments, dimeric NKG2D-Fc chimeras useful for the methodsdescribed herein may further comprise one or more accessory moieties,such as a tag sequence and a signal sequence. For example, a tagsequence can be used for detecting and/or isolating the polypeptide.Examples of tags include, without limitation: HA, Flag, Myc, Glu, Hisand Maltose basic protein. The tag sequence may be located at the aminoterminus, carboxyl terminus, or located somewhere in the middle of thedimeric NKG2D-Fc chimeric molecule (e.g., between modular peptidefragments), provided that the presence of such a tag does not interferewith the function of the dimeric NKG2D-Fc molecule. In some cases, a tagsequence is cleavable.

In some embodiments, dimeric NKG2D-Fc chimeras may optionally comprise asignal sequence. A signal sequence is a short (typically about 3-60amino acids long) peptide chain that directs the post-translationaltransport of a polypeptide, thereby allowing a greater yield of thepolypeptide. The amino acid sequences of a signal sequence directpolypeptides (which are synthesized in the cytosol) to certainsubcellular compartments, e.g., organelles. A signal sequence is alsoreferred to as a targeting signal, a signal peptide, a transit peptide,or a localization signal. In some embodiments, a signal sequence iscleaved from the polypeptide by signal peptidase after the polypeptideis transported.

In some embodiments, the dimeric NKG2D chimera contains an N-terminalmodified IL-2 signal sequence, which allows for optimal expression andsecretion of NKG2D-Fc construct. See, for example, Zhang et al., 2004,J. Gene Med., 7:354-65. In some embodiments, the dimeric NKG2D chimeracontains a signal peptide derived from the polypeptide sequence of CD33.For example, the CD33 signal peptide may correspond to amino acidresidues 1-16 of the CD33 polypeptide sequence. One of ordinary skill inthe art will understand that there are a number of other suitable signalpeptide sequences that may be used to practice the methods provided inthis disclosure. In addition, where there is a signal peptide present inthe NKG2D chimera, extra amino acid residues, e.g., a spacer, may beoptionally inserted between the N-terminus signal peptide and the Fcportion of the chimera. In some embodiments, for example, a signalsequence is followed by a Met-Asp dipeptide spacer.

Preparation of Dimeric NKG2D-Fc

The art is familiar with molecular biological and biochemical techniquesfor preparing a dimeric NKG2D-Fc chimera with desired features.Preferably, dimeric NKG2D-Fc chimeric constructs are produced byconventional recombinatory DNA methods. In preferred embodiments, adimeric NKG2D-Fc chimera is produced as a single (e.g., contiguous)recombinant polypeptide. In other embodiments, two or more portions ofdimeric NKG2D-Fc are produced as separate fragments and are subsequentlylinked together to yield a dimeric NKG2D-Fc molecule. For example, eachNKG2D portion (e.g., NKG2D₁, NKG2D₂) of the chimera and an Fc portion ofthe dimeric NKG2D-Fc are each produced as separate recombinantpolypeptides then fused together by a chemical linking means to yielddimeric NKG2D-Fc. This production methodology may be preferredparticularly in situations where a non-peptide linking molecule isemployed. Similarly, this production methodology may be also preferredif a dimeric NKG2D-Fc chimera does not fold correctly (e.g., does notproperly bind a ligand) when made as a single contiguous polypeptide.

For the production of recombinant polypeptides, a variety of hostorganisms may be used. Suitable hosts include, but are not limited to:bacteria such as E. coli, yeast cells, insect cells, plant cells, andmammalian cells. Choice of a suitable host organism will depend on theparticular application of the dimeric NKG2D-Fc chimera. The skilledartisan will understand how to take into consideration certain criteriain selecting a suitable host for producing the recombinant polypeptide.Factors affecting selection of a suitable host include, for example,post-translational modifications, such as phosphorylation andglycosylation patterns, as well as technical factors, such as thegeneral expected yield and the ease of purification. Host-specificpost-translational modifications of a dimeric NKG2D-Fc, which is to beused in vivo, should be carefully considered because certainpost-specific modifications are known to be highly immunogenic(antigenic).

Once produced, dimeric NKG2D-Fc can be purified by any suitable means,such as chromatographic methods known to those of skill in the art.Examples of chromatographic methods include gel filtrationchromatography. See, for example, Caine et al., Protein Expr. Purif.,1996, 8:159-66. In some embodiments, dimeric NKG2D-Fc is purified byProtein A immunoaffinity chromatography.

As will be recognized by one of ordinary skill in the art, dimeric NKG2Dchimera portions also can be prepared and isolated separately, andjoined by chemical synthesis.

NKG2D Receptor Ligands

In any of the embodiments described in this disclosure, dimeric NKG2D-Fcis capable of binding the endogenous ligand of the NKG2D receptor. KnownNKG2D-ligands in humans include MICA, MICB, RAET-1G, ULBP1, ULBP2,ULBP3, ULBP4, ULBP5, and ULBP6. Preferably, the dimeric NKG2D-Fc chimeradescried in the present disclosure is capable of binding more than onetype of NKG2D receptor ligand.

In some embodiments, the dimeric NKG2D-Fc chimeric molecules bindligands with high affinity of 10⁻⁴ M or less, 10⁻⁷ M or less, or withsubnanomolar affinity, e.g., 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1nM or even less. In some embodiments, the binding affinity of thedimeric NKG2D-Fc molecule for its ligands is at least 5×10⁶ Ka, at least1×10⁷ Ka, at least 2×10⁷ Ka, at least 1×10⁸ Ka, or greater.

In some embodiments, NKG2D-Fc binds preferentially to (e.g., with higheraffinity for) a subset of NKG2D receptor ligands. 3D structural data incombination with mutagenesis analyses have revealed that NKG2D ispermissive in the recognition and binding of a diverse array of itsendogenous ligands.

A ligand for NKG2D may be expressed on a cell surface. Alternatively, aligand for NKG2D may be “shed” from the cell surface and is present as asoluble ligand. It has been known in certain cancers that NKG2D ligandssuch as MICA are over-expressed and in some cases released (e.g., shed)into the bloodstream or surrounding tissues in a soluble form, e.g., insera. It is believed that this contributes, at least in part, to thepathogenesis and/or progression of cancer. Thus, the dimeric NKG2D-Fc isuseful for binding such ligand, either present on cell surface or as areleased form, in counterbalancing the expression of the ligands thatare present at an abnormally elevated level by functioning as aneutralizing agent.

Where an NKG2D ligand is expressed on the surface of cancer cells of asubject, dimeric NKG2D-Fc described in the present disclosure binds tothe cell surface ligand when administered to the subject. The binding ofthe dimeric NKG2D-Fc chimera to its ligand may prevent activation ofendogenous NKG2D receptors present on NK cells. Where an NKG2D ligand is“shed” from cancer cells, e.g., released into the bloodstream of asubject, dimeric NKG2D-Fc described herein binds to the soluble ligand,sequestering it from further action.

Therapeutic Applications

Normally, expression of the NKG2D ligands appears to be confined to thegastrointestinal epithelium. Little expression is observed in quiescentepithelial cells, but higher levels of expression occur in rapidlyproliferating cells. Expression of the NKG2D ligands is alsoup-regulated in various transformed cells, particularly those ofepithelial origin. Accordingly, provided herein are methods for treatingcancer or symptoms of cancer in a subject. The methods compriseadministering to the subject a therapeutically effective amount ofdimeric NKG2D-Fc that binds NKG2D ligands in vivo.

The terms “treating,” “treatment,” and “treat” and the like in thecontext of a cancer therapy refer to the administration of a compositioncomprising dimeric NKG2D-Fc as described herein to a subject who hascancer. The composition is administered to the subject in an amount thatis therapeutically effective. As used herein, a therapeuticallyeffective amount refers to an amount of the therapeutic that is believedto effectuate a beneficial effect with statistical significance on thesubject having the disease or disorder, such as certain types of cancer.Generally, a therapeutically effective amount is determined byadministering the composition to a population of subjects with specifiedconditions (such as progression or stage of a disease) and evaluatingthe outcome in response. As used herein, therapeutic treatment shallinclude, for example, complete prevention or abolishment of the symptomsof a disease, a delay in onset of the symptoms of a disease, orlessening in the severity of a disease.

Cancer

Dimeric NKG2D-Fc chimeras are believed to be broadly useful forimmunotherapy for a wide variety of cancers, where the expression of oneor more NKG2D ligands is elevated in a subject. Cancer broadly refers toa proliferative disease involving transformed cells, including bothpre-malignant and malignant disorders. The present invention is usefulfor treating a subject having cancer that is characterized byover-expression of one or more NKG2D ligands. In some embodiments, thecancer is characterized by over-expression of one (or predominantly one)ligand of the NKG2D receptor. In other embodiments, the cancer ischaracterized by over-expression of two or more NKG2D ligands.

The methods disclosed herein are useful therapeutics for the treatmentof pre-malignant disorders that carry with them a risk of progressing tomalignancy.

Examples of such disorders include, without limitation, dysplasia,hyperplasia, and plasma cell disorders such as monoclonal gammopathy ofundetermined significance (MGUS) and smoldering multiple myeloma (SMM).In some embodiments, the cancer is melanoma, lung, breast, kidney,ovarian, prostate, pancreatic, gastric, and colon carcinoma, lymphoma orleukemia. In some embodiments, the cancer is melanoma. In someembodiments, the cancer is a plasma cell malignancy, for example,multiple myeloma (MM) or pre-malignant condition of plasma cells. Insome embodiments, the cancer is melanoma, lung cancer, plasma cellcancer, leukemia, lymphoma, ovarian cancer, colon cancer, pancreaticcancer or prostate cancer. In some embodiments, the subject has beendiagnosed as having a cancer or as being predisposed to cancer. Thus,methods disclosed herein are also useful to treat a subject who has hada metastasis and is therefore susceptible to a relapse or recurrence.The methods are particularly useful in high-risk individuals who, forexample, have a family history of cancer or metastasizing tumors, orshow a genetic predispositions for a cancer metastasis. Specifically,the methods are directed to treating cancer that is associated withNKG2D ligand expression. In some embodiments, an NKG2D ligand is MICA.Thus, in some embodiments, the cancer causes MICA-related tumors.

Whether a particular subject (e.g., patient) should receive a cancertherapy comprising NKG2D-Fc can be determined by testing for aberrantexpression of one or more NKG2D ligands in the subject. “Aberrantexpression of one or more NKG2D ligands” in the subject meansover-expression of the ligand(s) in a biological sample obtained fromthe subject. In some embodiments, a biological sample may include abiopsy sample taken from a tissue of the subject suspected to becancerous. For example, in some cases, a biological sample is collectedfrom a solid tumor to test for malignancy. In other cases, a biologicalsample may constitute a blood sample, e.g., serum, a stool sample, urinesample, etc. A biological sample may be any cell or tissue sample thatis collected from a subject for the purpose of testing for the diagnosisor progression of a disease, such as cancer.

One of ordinary skill in the art is familiar with a variety oflaboratory techniques and protocols used to assay for the presence ofand the levels of one or more markers present in a biological sample. Todetermine whether a subject has cancer that is associated withover-expression of NKG2D ligand(s), typically immunoaffinity assays areperformed. In certain situations, depending on the type of biologicalsamples that are available, immunohistological or immunocytochemicalanalyses may be carried out. A number of antibodies are commerciallyavailable for performing these analyses. Methods commonly employed forthis purpose include, but are not limited to, ELISA, immunoblotting, andimmunohistochemistry.

Subjects

The methods disclosed herein can be applied to a wide range of species,e.g., humans, non-human primates (e.g., monkeys), horses, cattle, pigs,sheep, deer, elk, goats, dogs, cats, rabbits, guinea pigs, hamsters,rats, and mice, which are known to develop cancer. Thus, a “subject” asused herein is a mammalian subject having a disease, or at risk ofdeveloping a disease associated with an abnormal expression of at leastone NKG2D ligand, such as cancer. In preferred embodiments, the subjectis a human subject having a cancer presenting elevated levels of one ormore NKG2D ligands. In some embodiments, the NKG2D ligands include MICA.

If a subject has been shown to express an elevated level of one or moreNKG2D ligands, the subject may be treated with the methods describedherein. In some circumstances, a subject has received or is receivinganother cancer therapy. In some embodiments, the cancer may be inremission. In some cases, the subject is at risk of having recurrence,e.g., metastasis. In some embodiments, the over-expression of one ormore NKG2D ligands is limited to cancerous cells, e.g., tumors. In someembodiments, at least one of the NKG2D ligands expressed by cancer cellsare shed into the blood stream, and thus detectable in the serum of thesubject. Depending on the phenotype of a particular cancer, it may bepossible to target one or more ligands which are over-expressed(expressed by tumor cells) over the other ligands, whose expression isnot significantly affected.

Modes of Action

The instant invention is based, in part, on the surprising discoverythat that a chimeric molecule comprising two NKG2D fragments and an Fcfragment (e.g., a dimeric NKG2D-Fc chimera), which is capable of bindingone or more NKG2D ligands, induces tumor cell death with improvedefficacy compared to chimeric molecules comprising a single NKG2Dfragment and an Fc fragment (e.g., a monomeric NKG2D-Fc chimera).

Without being limited by any particular theory, it appears that dimericNKG2D-Fc chimeras can function through the two major components of theimmune system: innate immunity and adaptive immunity. As used herein,innate immunity or the innate immune system refers to non-specific hostdefense mechanisms against foreign pathogens. Innate immunity includesboth physical barriers (e.g., skin, gastric acid, mucus or tears, aswell as cells) and active mechanisms such as NK cells, phagocytes andthe complement system. NK cells represent a major component of theinnate immune system. NK cells are cytotoxic, e.g., are able to attackcells that have been infected by microbes, as well as some kinds oftumor cells. The cytotoxic activity of NK cells is mediated throughcell-surface receptors that recognize MHC class I alleles. A number ofreceptor types are known in the art, including NKG2D, which is onereceptor subtype. Phagocytic cells include neutrophils, monocytes,macrophages, basophils and eosinophils. The complement system is abiochemical cascade of the immune system that helps clear pathogens froma host organism.

In general, adaptive immunity or the adaptive immune system refers to anantigen-specific antibody-mediated immune response. Adaptive immunity isgenerally mediated via specific antibody production by B lymphocytes andantigen-specific activity of T lymphocytes. The humoral responsemediated by B lymphocytes defends primarily against extracellularpathogens through the production of circulating antibodies that markforeign cells and molecules for destruction by other specialized cellsand proteins. The cellular response mediated by T lymphocytes defendspredominantly against intracellular pathogens and cancer cells bydirectly binding to and destroying the affected cells. According to thepresent disclosure, dimeric NKG2D-Fc, which is a non-antibody molecule,is believed to functionally mimic what is ordinarily the function ofspecific antibodies.

The present invention thus contemplates methods for cancer treatment,wherein dimeric NKG2D-Fc binds directly to tumor cells that areexpressing NKG2D ligands on the cell surface. In this mode of action,dimeric NKG2D-Fc can specifically identify for destruction tumor cellsthat over-express NKG2D ligands, but not healthy cells that do not.

Dimeric NKG2D-Fc can target any or all NKG2D ligands that are expressedon human tumor cells in at least two ways. One mechanism of mediatingtumor cell destruction is through the process of complement lysis (alsoreferred to as complement dependent lysis, complement-dependentcytotoxicity or CDC). A second way of mediating tumor cell destructionis by triggering antibody dependent cellular cytotoxicity (ADCC).

In some embodiments, dimeric NKG2D-Fc acts as an opsonizing agent.Opsonization is the process where cells or particles become coated withmolecules which allow them to bind to receptors on other cells, such asdendritic cells or phagocytes, to promote the uptake. Forantigen-presenting cells such as dendritic cells and macrophages,opsonization promotes efficient processing and presentation of antigens.Opsonizing agents that are capable of specifically binding to both thetarget (e.g., ligands) and particular receptors on antigen-presentingcells (e.g., FcRs) that can mediate internalization and subsequentantigen processing are particularly useful.

Tumor cells that express one or more ligands of the NKG2D receptor onthe cell surface can become opsonized, e.g., coated, with dimericNKG2D-Fc molecules. For example, the NKG2D portion of the chimera canbind to the ligands on the tumor cell surface, while leaving the Fcportion of the chimera exposed. Dendritic cells have FcγRs and thereforecan bind to and internalize the tumor antigen (e.g., NKG2D ligands),which then results in antigen presentation to cytotoxic T cells, alsoknown as CD8+ T cells. This is referred to as cross-priming. Similarly,opsonization results in the generation of MHC class II-restricted CD4+ Tcell responses. Through opsonization, therefore, the NKG2D-Fc chimeracan promote efficient cross-presentation (e.g., priming) by dendriticcells, leading to the induction of potent T cell responses against thetumor.

Cancer patients often suffer from immune suppression. In some cases, itis believed that the immune suppression, at least in part, may be causedby impaired NKG2D receptor signaling. Based on a prevailing model, forexample, shed MICA impairs host defense by inducing the internalizationof NKG2D receptor molecules on lymphocytes. Thus, according to thismodel, tumor cell shedding of MICA results in immune suppression throughdown-regulation of NKG2D surface expression.

Therefore, the methods provided herein are useful for counteracting orrelieving immune suppression by administering a composition comprisingdimeric NKG2D-Fc, particularly in situations where a patient exhibitselevated levels of soluble (i.e., shed) NKG2D ligand or ligands that aredetectable in sera. The mode of action is that NKG2D-Fc administered tothe patient binds to (thus sequestering) excess soluble ligands of NKG2Dthat were shed from tumors, thereby reversing the down-expression ofNKG2D receptors on cell surface that led to immune suppression.

Thus, the dimeric NKG2D-Fc chimera can have multiple therapeuticfunctions, including neutralizing soluble ligands that are shed by tumorcells, promoting ADCC and/or CDC in tumor cells expressing the cellsurface ligands and mediating cross presentation and priming of theadaptive immune system, including CD8 cytotoxic T-lymphocytes (CTLs) andtumor-specific antibody producing B-cells.

Administration

The dimeric NKG2D-Fc composition can be administered directly to asubject. The subject is preferably a mammal. The terms “administration”and “administer” refer to a means of providing a pharmaceutical agent toa subject such that the pharmaceutical agent is to contact its targetcells, e.g., cancer cells, in vivo, i.e., in the body of the subject. Insome embodiments, the composition comprising NKG2D-Fc is systematicallyadministered to a subject. In preferred embodiments, a systematicadministration is delivered via an intravenous injection. In someembodiments, the composition comprising dimeric NKG2D-Fc is administeredlocally. For example, in some cases, the composition may be delivereddirectly to or within close proximity of a solid tumor.

Pharmaceutically-Acceptable Carriers

In some aspects, the disclosure provides a composition comprising thedimeric NKG2D-Fc chimera as described by this document and apharmaceutically acceptable carrier. Generally, the compositioncomprising dimeric NKG2D-Fc can be suspended in apharmaceutically-acceptable carrier (e.g., physiological saline). Suchcarriers can include, without limitation, sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsinclude mineral oil, propylene glycol, polyethylene glycol, vegetableoils, and injectable organic esters, for example. Aqueous earnersinclude, without limitation, water, alcohol, saline, and bufferedsolutions. Preservatives, flavorings, and other additives such as, forexample, antimicrobials, anti-oxidants, chelating agents, inert gases,and the like also may be present. It will be appreciated that anymaterial described herein that is to be administered to a mammal cancontain one or more pharmaceutically acceptable carriers.

Routes of Administration

Any composition described herein can be administered to any part of thesubject's body via various administration routes. The composition can beadministered by intravenous, intraperitoneal, intramuscular,subcutaneous, intramuscular, intrarectal, intravaginal, intrathecal,intratracheal, intradermal, or transdermal injection, by oral or nasaladministration, by inhalation, or by gradual perfusion over time. Thecomposition can be delivered to specific tissue. For example, thecomposition can be delivered to, without limitation, the joints, nasalmucosa, blood, lungs, intestines, muscle tissues, skin, or peritonealcavity of a mammal. In a further example, an aerosol preparation of acomposition can be given to a subject by inhalation.

Dosage

The dosage required depends on the route of administration, the natureof the formulation, the nature of the patient's illness, the subject'ssize, weight, surface area, age, and sex, other drugs beingadministered, and the judgment of the attending physician. Suitabledosages are typically in the range of 0.01-1,000 μg/kg. Wide variationsin the needed dosage are to be expected in view of the variety ofdimeric NKG2D-Fc compositions available and the differing efficienciesof various routes of administration. Variations in these dosage levelscan be adjusted using standard empirical routines for optimization as iswell understood in the art. Administrations can be single or multiple(e.g., 2- or 3-, 4-, 6-, 8-, 10-, 20-, 50-, 100, 150-, or more fold).Encapsulation of the composition in a suitable delivery vehicle (e.g.,polymeric microparticles or implantable devices) may increase theefficiency of delivery.

Treatment Regimen

The duration of treatment with any composition provided herein can beany length of time from as short as one day to as long as the life spanof the host (e.g., many years). For example, dimeric NKG2D-Fccompositions can be administered once a month for three months, or oncea year for a period of ten years. It is also noted that the frequency oftreatment can be variable. For example, dimeric NKG2D-Fc compositionscan be administered once (or twice, three times, etc.) daily, weekly,monthly, or yearly. Dimeric NKG2D-Fc compositions can be administeredtogether, e.g., at the same point in time or sequentially, with one ormore other cancer therapies. For example, a patient can receive anautologous tumor cell vaccine followed by an anti-CTL4 antibody,followed by a dimeric NKG2D-Fc therapy, separated by intervals of hours,days, months or years.

Effective Amounts

An effective amount of any composition described herein can beadministered to a subject. The term “effective” as used herein refers toany amount that induces a desired therapeutic effect, such as an immuneresponse, while not inducing significant toxicity in the subject. Suchan amount can be determined by assessing a subject's biologicalreaction, e.g., immune response and improvement in a symptom, afteradministration of a known amount of a particular composition. Inaddition, the level of toxicity, if any, can be determined by assessinga subject's clinical symptoms before and after administering a knownamount of a particular composition. It is noted that the effectiveamount of a particular composition administered to a subject can beadjusted according to a desired outcome as well as the host's responseand level of toxicity. Significant toxicity can vary for each particularhost and depends on multiple factors including, without limitation, thesubject's disease state, age, and tolerance to pain.

Combination Therapy

In some embodiments, the subject in need of cancer treatment is treatedwith the dimeric NKG2D-Fc composition described herein in conjunctionwith additional cancer therapy. In some embodiments, the additionalcancer therapy includes a cytotoxic agent and/or non-cytotoxic agent. A“cytotoxic agent” refers to a substance that inhibits or prevents thefunction of cells and/or causes destruction of cells. The term isintended to include radioactive isotopes (e.g., ¹³¹I, ¹²⁵I, ⁹⁰Y and¹⁸⁶Re), chemotherapeutic agents, and toxins such as enzymatically activetoxins of bacterial, fungal, plant or animal origin or synthetic toxins,or fragments thereof. A non-cytotoxic agent refers to a substance thatdoes not inhibit or prevent the function of cells and/or does not causedestruction of cells. A “non-cytotoxic agent” may include an agent thatcan be activated to be cytotoxic. A non-cytotoxic agent may include abead, liposome, matrix or particle (see, e.g., U. S. Patent Publications2003/0028071 and 2003/0032995, which are incorporated by referenceherein). Such agents may be conjugated, coupled, linked or associatedwith a dimeric NKG2D-Fc composition described herein.

In some embodiments, conventional cancer medicaments are administeredwith the compositions described herein. In some cases, the subject inneed of cancer treatment is treated with the dimeric NKG2D-Fccomposition described herein in conjunction with one or more additionalagents directed to target cancer cells. Highly suitable agents includethose agents that promote DNA-damage, e.g., double stranded breaks incellular DNA, in cancer cells. Any form of DNA-damaging agent know tothose of skill in the art can be used. DNA damage can typically beproduced by radiation therapy and/or chemotherapy. DNA-damaging agentsare also referred to as genotoxic agents. As used herein, “inconjunction with” shall mean that dimeric NKG2D-Fc is administered to asubject concurrently with one or more additional therapies (eithersimultaneously or separately but in close proximity), prior to, or afteradministration of one or more additional therapies.

Examples of radiation therapy include, without limitation, externalradiation therapy and internal radiation therapy (also calledbrachytherapy) Energy sources for external radiation therapy includex-rays, gamma rays and particle beams, energy sources used in internalradiation include radioactive iodine (iodine¹²⁵ or iodine¹³¹),strontium⁸⁹, or radioisotopes of phosphorous, palladium, cesium, indium,phosphate, or cobalt. Methods of administering radiation therapy arewell known to those of skill in the art.

Examples of DNA-damaging chemotherapeutic agents that may beparticularly useful include, without limitation: Busulfan (Myleran),Carboplatin (Paraplatin), Carmustme (BCNU), Chlorambucil (Leukeran),Cisplatin (Platmol), Cyclophosphamide (Cytoxan, Neosar), Dacarbazme(DTIC-Dome), Ifosfamide (Ifex), Lomustme (CCNU), Mechlorethamme(nitrogen mustard, Mustargen), Melphalan (Alkeran), and Procarbazine(Matulane).

A number of other chemotherapeutic agents may be also used for themethod described herein, either alone or in combination. These include:methotrexate, vincristine, adriamycin, cisplatin, non-sugar containingchloroethylnitrosoureas, 5-fluorouracil, mitomycin C, bleomycin,doxorubicin, dacarbazine, taxol, fragyline, Meglamine GLA, valrubicin,carmustaine and poliferposan, MMI270, BAY 12-9566, RAS farnesyltransferase inhibitor, farnesyl transferase inhibitor, MMP,MTA/LY231514, LY264618/Lometexol, Glamolec, CI-994, TNP-470,Hycamtin/Topotecan, PKC412, Valspodar/PSC833, Novantrone/Mitroxantrone,Metaret/Suramin, Batimastat, E7070, BCH-4556, CS-682, 9-AC, AG3340,AG3433, Incel/VX-710, VX-853, ZD0101, ISI641, ODN 698, TA2516/Marmistat, BB2516/Marmistat, CDP 845, D2163, PD183805, DX8951f,Lemonal DP 2202, FK 317, Picibanil/OK-432, AD 32/Valrubicin,Metastron/strontium derivative, Temodal/Temozolomide, Evacet/liposomaldoxorubicin, Yewtaxan/Paclitaxel, Taxol/Paclitaxel, Xeload/Capecitabine,Furtulon/Doxifluridine, Cyclopax/oral paclitaxel, Oral Taxoid,SPU-077/Cisplatin, HMR 1275/Flavopiridol, CP-358 (774)/EGFR, CP-609(754)/RAS oncogene inhibitor, BMS-182751/oral platinum, UFT(Tegafur/Uracil), Ergamisol/Levamisole, Eniluracil/776C85/5FU enhancer,Campto/Levamisole, Camptosar/Irinotecan, Tumodex/Ralitrexed,Leustatin/Cladribine, Paxex/Paclitaxel, Doxil/liposomal doxorubicin,Caelyx/liposomal doxorubicin, Fludara/Fludarabine,Pharmarubicin/Epirubicin, DepoCyt, ZD1839, LU 79553/Bis-Naphtalimide, LU103793/Dolastain, Caetyx/liposomal doxorubicin, Gemzar/Gemcitabine, ZD0473/Anormed, YM 116, iodine seeds, CDK4 and CDK2 inhibitors, PARPinhibitors, D4809/Dexifosamide, Ifes/Mesnex/Ifosamide, Vumon/Teniposide,Paraplatin/Carboplatin, Plantinol/cisplatin, Vepeside/Etoposide, ZD9331, Taxotere/Docetaxel, prodrug of guanine arabinoside, Taxane Analog,nitrosoureas, alkylating agents such as melphelan and cyclophosphamide,Aminoglutethimide, Asparaginase, Busulfan, Carboplatin, Chlorombucil,cisplatin, Cytarabine HCl, Dactinomycin, Daunorubicin HCl, Estramustinephosphate sodium, Etoposide (VP16-213), Floxuridine, Fluorouracil(5-FU), Flutamide, Hydroxyurea (hydroxycarbamide), Ifosfamide,Interferon Alfa-2a, Alfa-2b, Leuprolide acetate (LHRH-releasing factoranalog), Lomustine (CCNU), Mechlorethamine HCl (nitrogen mustard),Mercaptopurine, Mesna, Mitotane (o.p′-DDD), Mitoxantrone HCl,Octreotide, Plicamycin, Procarbazine HCl, Streptozocin, Tamoxifencitrate, Thioguanine, Thiotepa, Vinblastine sulfate, Amsacrine (m-AMSA),Azacitidine, Erthropoietin, Hexamethylmelamine (HMM), Interleukin 2,Mitoguazone (methyl-GAG; methyl glyoxal bis-guanylhydrazone; MGBG),Pentostatin (2′deoxycoformycin), Semustine (methyl-CCNU), Teniposide(VM-26), and Vindesine sulfate, but it is not so limited.

In addition, the following agents may be also useful for the instantinvention: alkylating agents, such as carboplatin and cisplatin,nitrogen mustard alkylating agents, nitrosourea alkylating agents, suchas carmustine (BCNU), antimetabolites, such as methotrexate, folinicacid, purine analog antimetabolites, mercaptopurine, pyrimidine analogantimetabolites, such as fluorouracil (5-FU) and gemcitabine (Gemzar®),hormonal antineoplastics, such as goserelin, leuprolide, and tamoxifen,natural antineoplastics, such as aldesleukin, interleukin-2, docetaxel,etoposide (VP-16), interferon alfa, paclitaxel (Taxol®), and tretinoin(ATRA), antibiotic natural antineoplastics, such as bleomycin,dactmomycin, daunorubicin, doxorubicin, daunomycin and mitomycinsincluding mitomycin C, and vinca alkaloid natural antineoplastics, suchas vinblastine, vincristine, vindesine, hydroxyurea, acetone,adriamycin, ifosfamide, enocitabine, epitiostanol, aclarubicin,ancitabine, nimustine, procarbazine hydrochloride, carboquone,carboplatin, carmofur, chromomycin A3, antitumor polysaccharides,antitumor platelet factors, cyclophosphamide (Cytoxan®), Schizophyllan,cytarabine (cytosine arabinoside), dacarbazine, thiomosine, thiotepa,tegafur, dolastatins, dolastatin analogs such as auristatin, CPT-11(irinotecan), mitozantrone, vinorelbine, teniposide, aminopterin,carbomycin, esperamicins (See, e.g., U.S. Pat. No. 4,675,187, which isincorporated by reference herein), neocarzinostatin, OK 432, bleomycin,furtulon, broxundine, busulfan, honvan, peplomycin, bestatin(Ubenimex®), interferon-β, mepitiostane, mitobromtol, melphalan, lamininpeptides, lentinan, Coriolus versicolor extract, tegafur/uracil,estramustine (estrogen/mechlorethamine), thalidomide, and lenalidomide(Revlmid®).

Other suitable chemotherapeutics include proteasome inhibiting agents.Proteasome inhibitors block the action of proteasomes, cellularcomplexes that degrade proteins, particularly those short-lived proteinsthat are involved in cell maintenance, growth, division, and cell death.Examples of proteasome inhibitors include bortezomib (Velcade®),lactacystin (AG Scientific, Inc, San Diego, CA), MG132 (BiomolInternational, Plymouth Meeting, PA) PS-519, eponemycin, epoxomycin,aclacinomycin A, the dipeptide benzamide, CVT-63417, and vinyl sulfonetripeptide proteasome inhibitors.

In some embodiments, the methods described herein are used inconjunction with one or more other cancer treatments, including cancerimmunotherapy. Cancer immunotherapy is the use of the immune system toreject cancer. The main premise is stimulating the subject's immunesystem to attack the tumor cells that are responsible for the disease.This can be either through immunization of the subject, in which casethe subject's own immune system is rendered to recognize tumor cells astargets to be destroyed, or through the administration of therapeutics,such as antibodies, as drugs, in which case the subject's immune systemis recruited to destroy tumor cells by the therapeutic agents. Cancerimmunotherapy includes an antibody-based therapy and cytokine-basedtherapy.

A number of therapeutic monoclonal antibodies have been approved by theFDA for use in humans, and more are underway. The FDA-approvedmonoclonal antibodies for cancer immunotherapy include antibodiesagainst CD52, CD33, CD20, ErbB2, vascular endothelial growth factor andepidermal growth factor receptor. These and other antibodies targetingone or more cancer-associated antigen are thus suitable for use in acombination therapy to be administered in conjunction with dimericNKG2D-Fc. Examples of monoclonal antibodies approved by the FDA forcancer therapy include, without limitation: Rituximab (available asRituxan™), Trastuzumab (available as Herceptin™), Alemtuzumab (availableas Campath-IH™), Cetuximab (available as Erbitux™), Bevacizumab(available as Avastin™) Panitumumab (available as Vectibix™), Gemtuzumabozogamicin (available as Mylotarg™) Ibritumomab tiuxetan (available asZevalin™) and Tositumomab (available as Bexxar™) Examples of monoclonalantibodies currently undergoing human clinical testing for cancertherapy in the United States include, without limitation: WX-G250(available as Rencarex™) Ipilimumab (available as MDX-010), Zanolimumab(available as HuMax-CD4), Ofatunumab (available as HuMax-CD20), ch14.18,Zalutumumab (available as HuMax-EGFr), Oregovomab (available as B43.13,OvalRex™), Edrecolomab (available as IGN-101, Panorex™), 131I-chTNT-I/B(available as Cotara™), Pemtumomab (available as R-1549, Theragyn™),Lintuzumab (available as SGN-33), Labetuzumab (available as hMN14,CEAcide™) Catumaxomab (available as Removab™), CNTO 328 (available ascCLB8), 3F8, 177Lu-J591, Nimotuzumab, SGN-30, Ticilimumab (available asCP-675206), Daclizumab (available as Zenapax™), Epratuzumab (availableas hLL2, LymphoCide™), ⁹⁰Y-Epratuzumab, Galiximab (available asIDEC-114), MDX-060, CT-011, CS-1008, SGN-40, Mapatumumab (available asTRM-I), Apolizumab (available as HuID10, Remitogen™) and Volociximab(available as M200).

Cancer immunotherapy also includes a cytokine-based therapy. Thecytokine-based cancer therapy utilizes one or more cytokines thatmodulate a subject's immune response. Non-limiting examples of cytokinesuseful in cancer treatment include interferon-α (IFN-α), interleukin-2(IL-2), Granulocyte-macrophage colony-stimulating factor (GM-CSF) andinterleukin-12 (IL-12).

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, and copending patent applications) cited throughout this application arehereby expressly incorporated by reference.

EXAMPLES

Constructs

hIgG1

This construct is the hIgG1 portion from the parental pFUSE-hIgG1 vector(Invivogen).

hNKG2Dx2-hIgG1

Two copies of human NKG2D (F78-V216), with an amino acid spacer betweenthem, were cloned via restriction-free cloning 5′ of the pFUSE-hIgG1vector (Invivogen). A schematic of this construct is depicted in FIG. 1, left panel.

hNKG2D, RefSeq NP_031386.2 , amino acids 78-216: (SEQ ID NO: 1)FLNSLFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTP NTYICMQRTVhIgG1-X or hNKG2Dx2-hIgG1-X

Using either the hIgG1 vector or the hNKG2Dx2-hIgG1 parental vectors,described above, various constructs were cloned 3′ of the hIgG1 segment(denoted by “X” above). These constructs are described below.

hIL15/hIL15Ra

A codon optimized version of a portion of the human IL-15 receptor alpha(hIL15Ra, GenBank AAP69528.1, amino acids 31-107, was fused to a codonoptimized version of IL-15 (IL15, GenBank AAX37025, amino acids 22-135.The hIL15Ra and hIL-15 were separated by a twenty amino acid (G₄S)₄ (SEQID NO: 3) linker. Amino acid sequences are shown below.

hIL15Ra, GenBank AAP69528.1, amino acids 31-107: (SEQ ID NO: 8)ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPIL15, GenBank AAX37025, amino acids 22-135: (SEQ ID NO: 9)NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCK ECEELEEKNIKEFLQSFVHIVQMFINTSaPD1

The ScFv for anti-mouse PD1 was cloned 3′ of the hIgG1 segment in thehIgG1 vector or the hNKG2Dx2-hIgG1 vector.

aCTLA4

The ScFv for anti-mouse CTLA4 was cloned 3′ of the hIgG1 segment in thehIgG1 vector or the hNKG2Dx2-hIgG1 vector.

Heterologous Expression and Purification

The indicated fusion constructs were produced in 293FT cells by calciumphosphate transfection of plasmids encoding the constructs. Supernatantswere collected, and the fusion constructs were purified by Protein Achromatography. 1 μg of each construct (reduced with 2-mercaptoethanoland heated to 70° C. for 10 minutes or not) was loaded onto an 8-10%SDS-PAGE gel, run at 100V for 60-120 minutes, and proteins werevisualized with Coomassie Blue staining. FIG. 2 shows thathNKG2Dx2-hIgG1-hIL15/Ra is produced as a single fusion protein, and iseasily purified by protein A.

Proliferation Assay

Human NK cells were isolated from normal donors using RosetteSep(StemCell Technologies). NK cells were labeled with 5 μMcarboxyfluorescein succinimidyl ester (CFSE) (Invitrogen). Cells werethen cultured in RPMI+10% FBS with various dilutions of the IL-15constructs (hIgG1-hIL15/Ra or hNKG2Dx2-20AA-hIgG1-hIL15/Ra) or IL-15 for4 days, and CFSE dilution was measured using a FACSCanto (BD). FIG. 3provides results showing that incubation with hNKG2Dx2-hIgG1-IL15/Rapromotes proliferation of human NK cells similarly to incubation withIL-15.

Killing Assay

Human NK cells were isolated from normal donors using RosetteSep(StemCell Technologies), and frozen in BamBanker (Wako). NK cells werethawed, and allowed to recover overnight in RPMI+10% FBS+200 IU/mLhIL-2. Cells were then washed three times in PBS, and added to varioustumor targets previously labeled with CFSE (Invitrogen). Cells werecentrifuged for 1 minute at 1000 rpm, and co-cultured for 5 hours at 37degrees in a humidified CO2 incubator. After 4 hours, 7-aminoactinomycinD (7-AAD) was added, and tumor target cell death was analyzed.

FIG. 4 shows that hNKG2Dx2-hIgG1-IL15/Ra promotes potent killing ofmultiple cell lines, and is superior to hNKG2Dx2-hIgG1 in cell lineswith moderate ligand expression. Panel A shows that constructs do notpromote killing of the B16 tumor cell line, which does not expressNG2D-L. Both hNKG2Dx2-hIgG1 and hNKG2Dx2-hIgG1-IL15/Ra constructsequally promote killing of a synthetic B16 tumor cell line expressinghigh levels of NKG2D ligand (FIG. 4 , panel B). Various tumors expressdifferent levels of NKG2D ligands on their cell surface, as measured byNKG2D fusion protein binding (FIG. 4 , panel C). The hNKG2Dx2-hIgG1 andhNKG2Dx2-hIgG1-IL15/Ra constructs also promote killing of tumorsnaturally expressing high levels of NKG2D ligands (K562), but thehNKG2Dx2-hIgG1-IL15/Ra construct drives superior killing, in a gradedmanner inversely correlated with NKG2D ligand expression (FIG. 4 , panelD).

IFNγ ELISA

Human NK cells were isolated, frozen, and added to tumor targets asdescribed above, except that there was no overnight recovery period forthe NK cells (e.g., RPMI+10% FBS+200 IU/mL hIL-2), and tumor targetswere not labeled with CFSE. After 24 hours of co-culture, the plateswere spun down and supernatant was aspirated for analysis by IFN-γ ELISA(Becton Dickinson).

FIG. 5 shows that resting NK cells are activated by the fusion proteinto produce IFN-γ, but maximum production requires all three components:NKG2D, hIgG1, and IL-15. Note that N297Q is a mutation in hIgG1 thatprevents CD16 (expressed on NK) binding to hIgG1.

CD16 and IL-15 Activation

FIG. 6 shows that pre-activated NK cells (e.g., incubated overnight withhIL-2) require CD16 binding to kill target cells, but do not requireIL-15.

Human NK cells were isolated, frozen, and added to tumor targets, asdescribed above. There was no overnight recovery period for the NKcells. Tumor targets were labeled with CFSE. FIG. 7 shows that optimalactivation of, and killing by, resting NK cells requires CD16 bindingand IL-15 activation.

Improved Characteristics of Dimeric NKG2D-Fc Constructs

A protein model showing NKG2Dx2-hIgG1 in complex with the NKG2D ligandMICA is shown in FIG. 13 . Binding of NKG2Dx2-hIgG1 and hNKG2Dx1-hIgG1constructs to MICA*008 was assayed by ELISA and flow cytometry. FIG. 8shows ELISA data demonstrating that NKG2Dx2-hIgG1 binds to MICA*008 withimproved avidity as compared to hNKG2Dx1-hIgG1. FIG. 9 depicts flowcytometry data showing that hNKG2Dx2-hIgG1 binds with improved avidityto NKG2D ligand-expressing cells as compared to hNKG2Dx1-hIgG1.

FIG. 10 shows NKG2D-Fc drives NK cell killing of ligand-positive (e.g.,NKG2D ligand expressing) targets. In particular, hNKG2Dx2-20AA-hIgG1mediated significantly higher killing of B16 cells thanhNKG2Dx1-20AA-hIgG1.

FIG. 11 shows hNKG2Dx2-hIgG1 improves killing of NKG2D ligand-expressingcells as compared to hNKG2Dx1-hIgG1. B16-ULBP20E and HeyA8 cells weretested.

Tumors can shed their NKG2D ligands, which further impairs the immuneresponse. One potential solution to this issue is to “sponge up” solubleNKG2D ligands (e.g., soluble MICA) to restore immune system function.FIG. 12 shows NKG2Dx2-hIgG1 neutralization of soluble MICA is superiorto NKG2Dx1-hIgG1.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method of treating an NKG2D ligand expressingcancer, comprising administering to a subject in need thereof apolypeptide chain comprising: (a) a first ligand binding fragment ofhuman NKG2D; (b) a second ligand binding fragment of human NKG2D; and(c) an Fc domain of a human immunoglobulin comprising: (i) a CH2 domain;and (ii) a CH3 domain.
 2. The method of claim 1, wherein the first andsecond ligand binding fragments each comprise an extracellular fragmentof the NKG2D receptor.
 3. The method of claim 2, wherein the first andsecond ligand binding fragments are identical.
 4. The method of claim 2,wherein the first and second ligand binding fragments are different. 5.The method of claim 1, wherein the human immunoglobulin is an IgG1. 6.The method of claim 1, wherein the polypeptide chain comprises a linkerbetween the first and second ligand binding fragments.
 7. The method ofclaim 6, wherein the linker is a flexible peptide linker.
 8. The methodof claim 7, wherein the linker comprises two or more of the followingamino acids: glycine, serine, alanine, and threonine.
 9. The method ofclaim 7, wherein the linker is 25 or fewer amino acids in length. 10.The method of claim 7, wherein the linker is 20 or fewer amino acids inlength.
 11. The method of claim 7, wherein the linker is 12 or feweramino acids in length.
 12. The method of claim 1, wherein the Fc domainof the polypeptide chain further comprises a hinge domain.
 13. Themethod of claim 1, wherein the polypeptide chain does not comprise acytokine.
 14. The method of claim 1, wherein the polypeptide chain doesnot comprise a drug moiety.
 15. The method of claim 1, wherein thepolypeptide chain administered is in the form of a homodimer comprisingtwo individual polypeptide chains dimerized through their respective Fcdomains.
 16. The method of claim 1, wherein the NKG2D ligand expressingcancer is melanoma, lung cancer, plasma cell cancer, leukemia, lymphoma,ovarian cancer, colon cancer, pancreatic cancer or prostate cancer. 17.The method of claim 15, wherein the NKG2D ligand expressing cancer ismelanoma, lung cancer, plasma cell cancer, leukemia, lymphoma, ovariancancer, colon cancer, pancreatic cancer or prostate cancer.